EP2343535B1 - Fluoreszenzmarkierungsmaterial und fluoreszenzmarkierungsmittel - Google Patents

Fluoreszenzmarkierungsmaterial und fluoreszenzmarkierungsmittel Download PDF

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Publication number
EP2343535B1
EP2343535B1 EP09823469.3A EP09823469A EP2343535B1 EP 2343535 B1 EP2343535 B1 EP 2343535B1 EP 09823469 A EP09823469 A EP 09823469A EP 2343535 B1 EP2343535 B1 EP 2343535B1
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Prior art keywords
fluorescent labeling
zinc oxide
zhie
fluorescent
labeling agent
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French (fr)
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EP2343535A4 (de
EP2343535A1 (de
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Moriyuki Sato
Morihiko Nakamura
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Shimane University NUC
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Shimane University NUC
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    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/54—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing zinc or cadmium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T428/00—Stock material or miscellaneous articles
    • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
    • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]
    • Y10T428/2991—Coated

Definitions

  • the present invention relates to a fluorescent labeling material and a fluorescent labeling agent, in particular, a fluorescent labeling material using a material free of toxicity on living organisms and a fluorescent labeling agent using the fluorescent labeling material.
  • CdSe cadmium selenide
  • ZnO zinc oxide
  • Both of CdSe and zinc oxide are suitable as light emission sources because: CdSe can serve as a quantum dot to develop a desired color and has a sharp spectrum; and zinc oxide is a material free of toxicity on living organisms.
  • US 2008/0020483 A1 provides a fluorescence detecting method, comprising irradiating an excitation light in the UV range on a sample containing a nanoparticle fluorescent material having an emission half-value width of 50 nm to 200 nm as the fluorescent material and detecting the fluorescence emitting from the sample in the visible range.
  • US 2006/0086925 A1 describes a nanoparticle fluorescent material of metal oxide or metal sulfide which is surface modified by a surface modification agent, wherein a half width of emission spectrum is 50 to 200 nm, and the surface modification agent is a compound represented by formula (I) or a degradation product thereof, and a dispersion of the same.
  • Formula (I) being M-(R) 4 and in formula (I), M is an Si or Ti atom, and R is an organic group.
  • the R groups may be the same or different from each other, but at least one R group is a group having reactivity with an affinity molecule.
  • WO 2007/078297 A2 relates to articles and methods involving luminescent films which may be useful in various applications.
  • Luminescent films of the present invention may comprise a layer of metal oxide nanoparticles and, in some cases, may interact with an analyte to generate a detectable signal, whereby the presence and/or amount of analyte can be determined.
  • fluorescence resonance energy transfer FRET
  • Such articles and methods may be useful in, for example, biological assays or in sensors.
  • WO 2008/066138 A1 discloses the use of a zinc oxide nanoparticle as an exciton-producing light source in vivo or in vitro. Also disclosed is a fluorescent labeling agent comprising a zinc oxide nanoparticle and a substance capable of selectively binding a targetin vivo or in vitro, wherein the substance is bound to the zinc oxide nanoparticle via at least one binder.
  • a fluorescent labeling agent comprising a zinc oxide nanoparticle having the surface coated with a coating film selected from ZnS, Mg x Zn 1-x O (provided that 0 ⁇ x ⁇ 1) and SiO 2 and a substance capable of selectively binding a target in vivo or in vitro, wherein the substance is bound to the zinc oxide nanoparticle via at least one binder.
  • CdSe is an excellent light emission source indeed, but involves the following problem owing to its toxicity on living organisms. Conditions under which CdSe is used and objects of labeling are limited.
  • a light emission source Upon labeling of a living organism, a light emission source must be caused to selectively adhere to the target. In this case, there arises a need for modifying a nanoparticle serving as the light emission source with an antibody or the like through a proper binder.
  • the binder as a modifying material is preferably organic matter from the viewpoint of a binding reaction because the kinds of targets, and by extension, the kinds of antibodies and the like are various. Meanwhile, good crystallinity is needed when zinc oxide is considered to be a fluorescent light-emitting material.
  • inorganic crystals including, but not limited to, zinc oxide each involve the following problem.
  • the more regular the crystal structure of any such inorganic crystal the higher the difficulty with which the surface of the crystal is modified or bound with organic matter.
  • surface-modified inorganic matter is so instable that it is difficult to develop a fluorescent labeling agent which brings together stability and light-emitting property.
  • the present invention has been made in view of the foregoing, and an object of the present invention is to provide a fluorescent labeling material and a fluorescent labeling agent each of which realizes stable fluorescent labeling of a living organism while using an inorganic crystal free of toxicity on living organisms as a light emission source.
  • a fluorescent labeling material according to claim 1 includes zinc oxide nanoparticles each surface-modified with an organic compound having an amino group placed at an outer end thereof, in which the organic compound includes a binding chain represented by the following formula.
  • various substances such as an antibody capable of selectively binding to a target to be fluorescently labeled can each be easily bound to a particle side through an amino group while a light emission source free of toxicity on living organisms is used.
  • an improvement in color development efficiency or luminous efficiency can be expected because an amide group and a urethane group each serving as an auxochrome exist so as to be adjacent to each other.
  • a stable light emission source having a strong bond can be obtained by simultaneously performing the formation of nanoparticles and surface modification by means of zinc acetate as a starting material. As a result, the number of objects to be fluorescently labeled can be increased.
  • the term “outer end” refers to a side opposite to the side on which the substance is bound to the particle, i.e., the outside when viewed from the particle.
  • the term “zinc oxide nanoparticle” as used herein can be interpreted as a zinc oxide nanocrystal in a sense that the nanoparticle must have crystallinity to such an extent as to emit fluorescence.
  • each molecule of the organic compound and each of the zinc oxide nanoparticles are not bound to each other at a ratio of 1:1, but instead a plurality of molecules of the organic compound are bound so as to cover the surface of each of the zinc oxide nanoparticles.
  • n is set equal to 1 to 6 here.
  • the compound has so good solubility in water as to be suitable for biological labeling.
  • n exceeds 6, the handleability of the compound deteriorates.
  • electrons are each requested to transfer by up to about 50 nm from the viewpoint of light-emitting property.
  • an upper limit for n is 6. It should be noted that n more preferably equals 1 to 4.
  • a fluorescent labeling material according to claim 2 is a fluorescent labeling material according to claim 1, in which the fluorescent labeling material has a particle diameter of 15 nm or less.
  • the invention according to claim 2 enables the fluorescent labeling of minute biological tissues or biological substances including targets in capillaries.
  • zinc oxide crystal particles each having a particle diameter of the order of single nanometers to about ten or so nanometers can be obtained by a sol-gel method or the like.
  • a fluorescent labeling material according to claim 3 is a fluorescent labeling material according to any one of claims 1 or 2, in which the zinc oxide nanoparticles each have crystallinity with which the nanoparticle emits fluorescence.
  • the invention according to claim 3 enables labeling by utilizing a light-emitting characteristic originating from the so-called crystallinity.
  • Light having any wavelength may be used as excitation light as long as the wavelength falls within such a region as to be free of any particular influence on biological labeling. Excitation may be performed with ultraviolet light, and visible light is also permitted in some cases.
  • a light source is, for example, a pulse laser such as helium-cadmium laser (325 nm) capable of continuous oscillation or nitrogen laser (having a wavelength of 337 nm), or an ultraviolet lamp such as a mercury lamp. It should be noted that upon observation, the fluorescence can be observed as light emission having a predetermined color such as a blue, green, orange, or red color through a filter as appropriate.
  • a fluorescent labeling agent according to claim 4 is a fluorescent labeling agent to be used in vivo or in vitro, including the fluorescent labeling material according to any one 3 of claims 1 to 3, in which: 3-[(2-aminoethyl)dithio]propionic acid (AEDP), 4-(p-azidosalicylamido)butylamine (ASBA), 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC), or EDC to which N-hydroxysulfosuccinimide (Sulfo-NHS) is added is bound thereto through the amino group; and a substance capable of selectively binding to a target to be fluorescently labeled is linked thereto.
  • AEDP 3-[(2-aminoethyl)dithio]propionic acid
  • ASBA 4-(p-azidosalicylamido)butylamine
  • EDC 1-ethyl-3-[3-dimethyla
  • the invention according to claim 4 can provide a fluorescent labeling agent which: can easily bind to a particle side; and suitably binds not only to a substance capable of selectively binding to a target but also to a substance that can be phagocytosed by, for example, a macrophage to realize easy labeling. Sulfo-NHS is added as a stabilizer.
  • the procedure for binding is not particularly limited. That is, the fluorescent labeling material and EDC or the like may be bound to each other first before the substance capable of selectively binding to the target is linked. Alternatively, EDC or the like and the substance capable of selectively binding to the target may be linked to each other first before the fluorescent labeling material is bound.
  • a fluorescent labeling agent according to claim 5 is a fluorescent labeling agent according to claim 4, in which the substance capable of selectively binding to a target is an antibody, an enzyme, a lectin, or a nucleic acid.
  • the invention according to claim 5 enables easy recognition of a target disease or the like in a biopsy or the like.
  • nucleic acid naturally comprehends DNA and RNA.
  • Other examples of the substance capable of selectively binding to the target include physiologically active substances (such as a hormone, a cytokine, and a growth factor), receptors, glucides (carbohydrates), and lipids.
  • the invention according to claim 6 is a use of the fluorescent labeling agent according to claim 4 or 5, including using as a target a tumor cell, a leukemia cell, a virus-infected cell, or a normal cell, a protein, an enzyme, or a nucleic acid.
  • the invention according to claim 6 enables easy recognition of a tumor or the like.
  • tumor cell comprehends both benign and malignant cells.
  • the invention according to claim 9 enables easy recognition of various biological substances not only on a cell surface but also in a cell cytoplasm.
  • a fluorescent labeling material capable of easily binding each of various substances such as an antibody capable of selectively binding to a target to be fluorescently labeled to a particle side through an amino group while using zinc oxide free of toxicity on living organisms.
  • a stable fluorescent labeling agent based on the material can be prepared.
  • the present invention has the following advantages as well.
  • the present invention can be prepared with a commercially available reagent, and is excellent in industrial mass productivity (can be produced at a low cost).
  • FIGS. 18-1 , 18-3 , 19-1 , 20-1 , 21-1 , 22-1 , and 23-1 illustrate photographs in gray scale
  • FIGS. 18-2 , 18-4 , 19-2 , 20-2 , 21-2 , 22-2 , and 23-2 are views drawn on the basis of the above-mentioned respective views. It should be noted that in each of the views illustrated in gray scales, a contrast and brightness are adjusted as appropriate in order that clear display may be achieved. Views or photographs illustrated in gray scales are submitted in designated states as required.
  • Embodiment 1 the case where n in the chemical formula (1) equals 2 is described.
  • a fluorescent labeling material named ZHIE by the inventors of the present invention was prepared with a commercially available reagent at a low cost with facility is described.
  • the phagocytic action or the like of a mouse macrophage on zymosan is actually observed by preparing a fluorescent labeling agent with the fluorescent labeling material is described.
  • ZHIE is a fluorescent labeling material with extremely high general-purpose property is described by proving ZHIE free of toxicity.
  • a fluorescent labeling material of Embodiment 1 can be produced by the following method.
  • FIG. 1 illustrates the substance in such a manner that the surface of the zinc oxide nanocrystal is modified with one molecule of 3-hydroxypropionic acid, a large number of molecules of 3-hydroxypropionic acid are actually bound to the surface so as to form a coating.
  • the substance is represented as ZHP as appropriate.
  • FIG. 6 shows the fluorescent spectrum of ZHP in methanol.
  • ZHP was confirmed to emit green light.
  • FIG. 7 shows the peak wavelength of a fluorescent spectrum obtained by using ZHP prepared by altering the molar ratio. It should be noted that the figure shows a local maximum absorption wavelength at the same time.
  • ZHP has an OH group at an outer end thereof.
  • ZHI is prepared from ZHP by utilizing the OH group ( FIG. 2 ).
  • 0.5 Gram of ZHP (prepared at a ratio "ZnO:HPA" of 40:1) was added to 5 ml of N,N-dimethylacetamide (manufactured by Wako Pure Chemical Industries, Ltd.), and then the mixture was subj ected to an ultrasonic treatment so that ZHP might be dispersed well.
  • a ten-fold molar amount of ethyl acetate isocyanate ester manufactured by Tokyo Kagaku Kougyou Co., Ltd.
  • the powder was considered to be a zinc oxide nanocrystal whose surface was modified with a binding chain represented by the following formula (see FIG. 2 ).
  • the measurements of the FT-IR spectrum ( FIG. 8 ) and XRD pattern ( FIG. 9 ) of the powder confirmed that the zinc oxide nanocrystal whose surface was modified with the above-mentioned binding chain was prepared indeed.
  • FIG. 2 illustrates the substance in such a manner that the surface of the zinc oxide nanocrystal is modified with one molecule of the above-mentioned organic matter, a large number of molecules of the organic matter are actually bound to the surface so as to form a coating.
  • the substance is represented as ZHI as appropriate.
  • FIG. 10 the fluorescent spectrum of ZHI in methanol was measured.
  • ZHI was able to be confirmed to emit substantially green light.
  • the zinc oxide nanoparticles in ZHI each still had crystallinity with which the nanoparticle emitted fluorescence.
  • FIG. 11 shows the peak wavelength of the fluorescent spectrum of ZHI prepared by altering the molar ratio. It should be noted that the figure shows a local maximum absorption wavelength at the same time.
  • FIG. 12 shows the results of the measurement of the minimum reduction ratio of ZHI prepared from different molar ratios in a TD/DTA measurement.
  • the final reduction ratio reduces as the amount of the organic matter increases from 60:1 to 40:1, but the final reduction ratio remains nearly unchanged from 40:1 to 20:1.
  • a limit for the amount of the organic matter capable of binding to the surface of ZnO fell within the molar ratio range of about 1/40 to 1/50 with respect to ZnO.
  • ZHI had an ester group in the vicinity of its outside, the group was subjected to an amidation reaction so that an amide groupmight be introduced. In addition, an amino group was introduced into an outer end in order that binding property with an antibody or the like might be improved. That is, ZHIE was prepared from ZHI ( FIG. 3 ).
  • the powder was considered to be a zinc oxide nanocrystal whose surface was modified with a binding chain represented by the following formula (see FIG. 3 ).
  • Themeasurements of the FT-IR spectrum ( FIG. 13 ) and XRD pattern ( FIG. 14 ) of the powder confirmed that the zinc oxide nanocrystal whose surface was modified with the above-mentioned binding chain was prepared indeed. This is the binding chain in the case where n in the chemical formula (1) equals 2.
  • FIG. 3 illustrates the substance in such a manner that the surface of the zinc oxide nanocrystal is modified with one molecule of the above-mentioned organic matter having amino groups at the terminals, a large number of molecules of the organic matter are actually bound to the surface so as to form a coating.
  • the substance is represented as ZHIE as appropriate.
  • ZHIE fluorescent spectrum of ZHIE in methanol was measured.
  • ZHIE was able to be confirmed to emit substantially green light.
  • the zinc oxide nanoparticles in ZHIE each still had crystallinity with which the nanoparticle emitted fluorescence. Peaks are present at 412 nm and 532 nm, and are considered to be light emission based on an exciton and light emission based on a crystal defect, respectively.
  • FIG. 16 illustrates the result of the measurement of an absorption spectrum. A peak is observed at 348 nm, and is considered to be based on exciton absorption.
  • FIG. 17 shows the peak wavelength of the fluorescent spectrum of ZHIE prepared by altering the molar ratio. It should be noted that the figure shows a local maximum absorption wavelength at the same time.
  • FIGS. 18 show TEM photographs of ZHIE.
  • FIG. 18-1 is a photograph obtained by photographing a mass portion.
  • FIG. 18-3 is a photograph obtained by photographing dispersed small masses (part of which aggregate).
  • the small masses of ZHIE each have a size of the order of generally single nanometers, or specifically 5 nm to 6 nm.
  • particle diameters measured by dispersing ZHIE in methanol were each about several nanometers to fifteen nanometers in size, and had an average particle diameter of 11.7 nm. Accordingly, it was able to be confirmed that the nanoparticles were dispersed in the liquid alone or in a state of being an aggregate of several nanoparticles.
  • FIGS. 19 show the manner in which the dispersed solution emits light upon application of UV light from below the solution.
  • the foregoing preparation approach was able to provide a fluorescent labeling material of about ten or so nanometers or less in size, the fluorescent labeling material emitting fluorescence in a visible light region and having a surface modified with an organic substance having an amino group placed at an outer end thereof.
  • the material is excellent in mass productivity because the material can be prepared with a commercially available raw material.
  • ZHIE obtained by the former approach is more stable because a binding force between the organic matter and each of the zinc oxide nanoparticles is larger.
  • ZHIE described above is expected to have high luminous efficiency as well because an amide group and a urethane group each serving as an auxochrome exist and are adjacent to each other.
  • a fluorescent labeling agent to be used in vivo or in vitro was prepared with ZHIE. Described here is a method of preparing such a fluorescent labeling agent that zymosan is used as a substance capable of selectively binding to a target and the substance is bound to ZHIE through EDC.
  • HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
  • zymosan ⁇ glucan derived frombaker' s yeast: manufactured by Sigma-Aldrich Corporation
  • MES 2-(N-morpholino) ethanesulfonic acid
  • EDC 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride
  • sulfo-NHS manufactured by Pierce
  • the reaction was stopped by adding 2-mercaptoethanol (2-ME: manufactured by Wako Pure Chemical Industries, Ltd.) so that its final concentration might be 20 mM. Then, the resultant was subjected to centrifugal separation at 5000 rpm for 3 minutes so that zymosan might be precipitated. The supernatant was removed, and then 1 ml of an HEPES buffer solution was added to the remainder. The centrifugal separation operation was repeated twice so that 2-ME, and unreacted EDC and sulfo-NHS might be removed, and the buffer solution might be replaced with HEPES. It should be noted that those operations were quickly performed because EDC bound to zymosan was instable.
  • the fluorescent labeling agent 20 each show the manner in which fluorescence achieved by irradiating the fluorescent labeling agent with excitation light changed from ultraviolet light to visible light is observed as blue light emission, green light emission, or red light emission through a filter.
  • the fluorescent labeling agent enables the observation of fluorescence as various luminescent colors.
  • the fluorescent labeling agent was stable even after storage in the PBS at 4°C for one week.
  • Mouse macrophage-based cells Raw264. 7 (20, 000 cells/ml) were cultured in a 10% FBS-containing Dulbecco's modified Eagle's medium (DMEM: manufactured by GIBCO), and then zymosan to which zinc oxide nanoparticles were bound described above was added to the cells.
  • the addition amount was such that the number of molecules of zymosan was ten per cell. After a lapse of 30 minutes to 1 hour, the culture solution was replaced with a PBS.
  • the PBS was exchanged with a new PBS again. After a lapse of an additional three minutes, the PBS was removed, and then a 4% paraformaldehyde/PBS was added to the remainder so that the cells might be fixed for 15 minutes. The paraformaldehyde/PBS was removed, and then a 50% glycerol/PBS was added to the remainder. Then, the manner of fluorescence was observed.
  • the fluorescence was observed with a fluorescence microscope by using a high-pressure mercury lamp as excitation light.
  • An excitation wavelength ( ⁇ EX ) of 360 to 370 nm, a wavelength ( ⁇ DIC ) to be separated with a dichroic mirror of 400 nm, and a fluorescence wavelength ( ⁇ EM ) of 400 nm or more were observed.
  • FIGS. 21 are photographs showing the results of the observation. It was able to be confirmed that a zymosan portion taken up by the phagocytic action of each of the Raw264.7 cells was clearly labeled.
  • the phagocytic action of an alveolar macrophage was also observed. Specifically, the tail of an alive Balb/c mouse was subjected to intravenous injection with a fluorescent labeling agent (zymosan to which zinc oxide nanoparticles were bound). After a lapse of 30 minutes from the administration, the lung was taken out and sliced. The sliced lung was placed as it was on a slide glass, and then the manner of fluorescence was observed.
  • a fluorescent labeling agent zymosan to which zinc oxide nanoparticles were bound
  • the fluorescence was observed with a fluorescence microscope by using a high-pressure mercury lamp for excitation light.
  • An excitation wavelength ( ⁇ EX ) of 360 to 370 nm, a wavelength ( ⁇ DIC ) to be separated with a dichroic mirror of 400 nm, and a fluorescence wavelength ( ⁇ EM ) of 400 nm or more were observed.
  • FIGS. 22 are photographs showing the results of the observation. It was able to be confirmed that a zymosan portion taken up by the phagocytic action of the alveolar macrophage was clearly labeled. The result can be said to demonstrate that the fluorescent labeling agent stably flows in the blood in a living organism and does not lose its fluorescent characteristic.
  • the zinc oxide nanoparticles of which the fluorescent labeling agent obtained by the above-mentioned production method is constituted are a nontoxic material, show nearly no color deterioration, and as shown in FIGS. 20 , 21 , and 22 , enable dynamic observation of the phagocytic action of a target cell.
  • the fluorescent labeling agent can similarly label, for example, a cell membrane component derived from Escherichia coli as well as zymosan, and hence can find use in researches on phagocytosis and endocytosis.
  • ZHIE ZHIE was subjected to an ultrasonic treatment in methanol in the same manner as that described above so as to be dispersed. Then, the solvent was evaporated with a suction aspirator (provided that the evaporation treatment was terminated at such a level that the treated product did not completely exsiccate).
  • the cytotoxicity test of the fluorescent labeling material ZHIE of which the fluorescent labeling agent of the present invention was constituted was performed. Under a condition of the number of Raw264.7 cells of 50,000 cells/well, ZHIE was added at various concentrations (0 to 100 mM), and 24 hours after that, whether the cells were dead or alive was measured by a trypan blue method. FIG. 24 illustrates the results of the measurement. It was confirmed that the cells did not die out even at the highest concentration, i.e., 100 mM.
  • n in the chemical formula (1) equals 1 is described.
  • the case is such that glycolic acid is used instead of HPA as a raw material. Since glycolic acid is a solid crystal, glycolic acid is easy to handle as compared with HPA, and obviates the need for an operation such as moisture removal with an evaporator.
  • a fluorescent labeling material named ZGAI by the inventors of the present invention is described.
  • ZGAIE was able to be actually prepared by setting a molar ratio "ZnO:glycolic acid" to 40:1 or 80:1 in accordance with the above-mentioned schemes.
  • ZGAIE prepared at a molar ratio of 40:1 was used is described.
  • FIGS. 28, 29 , 30, and 31 illustrate the results of the measurements, respectively. Those results confirmed that the zinc oxide nanocrystal whose surface was modified with the binding chain obtained by setting n in the chemical formula (1) equal to 1 was prepared indeed.
  • fluorescent labeling materials in which n equals any one of 3, 4, 5, and 6 can be similarly obtained by using 4-hydroxybutyric acid, 5-hydroxyvaleric acid, 6-hydroxyhexanoic acid, and 7-hydroxyheptanoic acid as starting materials, respectively.
  • a desired fluorescent labeling agent can be designed with any such fluorescent labeling material by: binding EDC or the like through the amino group at the terminal of the material; and linking a substance capable of selectively binding to a target to be fluorescently labeled.
  • the zinc oxide nanoparticles of which the fluorescent labeling agent of the present invention is constituted can find use in assorted applications because the nanoparticles have the following characteristics.
  • the nanoparticles are a nontoxic material, show nearly no color deterioration, and can fluorescently label a target cell with ease.
  • a fluorescent labeling agent containing a biotin-labeled cancer-specific antibody and streptavidin for recognizing a cancer cell is conceivable.
  • An enhancing effect on fluorescence can be expected from streptavidin because streptavidin has such property as to capture biotin efficiently.
  • a fluorescent labeling agent prepared by binding ZHIE to a secondary antibody can be used as an indirect fluorescent antibody. That is, when the antibody of a mouse is used as a primary antibody, a fluorescent labeling agent serving as an anti-mouse antibody that emits fluorescence can be used as the secondary antibody.
  • the industrial usefulness of the secondary antibody is imponderable because the secondary antibody has general-purpose property in life science studies and is not fastidious about any antibody species.
  • the present invention can be used in a diagnosis for specifying a cancer portion during an operation.
  • a perioperative diagnosis for making an immediate judgment as to whether an affected area is positive or negative can be performed with a cancer antigen-specific antibody to which zinc oxide nanoparticles are bound by fluorescently labeling the affected area through the application of laser.
  • the present invention can be applied to a wide variety of clinical laboratory tests such as the diagnosis of a skin cancer because of its non-toxicity.

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Claims (6)

  1. Fluoreszenzmarkerstoff, der Zinkoxid-Nanopartikel umfasst, die jeweils mit einer organischen Verbindung mit einer Aminogruppe an ihrem äußeren Ende oberflächenmodifiziert sind, wobei die organische Verbindung eine Bindungskette umfasst, die durch folgende Formel dargestellt ist:
    Figure imgb0005
  2. Fluoreszenzmarkerstoff gemäß Anspruch 1, wobei der Fluoreszenzmarkerstoff einen Partikeldurchmesser von 15 nm oder weniger aufweist.
  3. Fluoreszenzmarkerstoff gemäß Anspruch 1 oder 2, wobei die Zinkoxid-Nanopartikel jeweils eine Kristallinität aufweisen, mit der die Nanopartikel Fluoreszenz emittieren.
  4. Fluoreszenzmarkerstoff zur Verwendung in vivo oder in vitro, umfassend den Fluoreszenzmarkerstoff gemäß mindestens einem der Ansprüche 1 bis 3, wobei:
    3-[(2-Aminoethyl)dithio]propionsäure (AEDP), 4-(p-Azidosalicylamido)butylamin (ASBA), 1-Ethyl-3-[3-dimethylaminopropyl]carbodiimidhydrochlorid (EDC) oder EDC, zu dem N-Hydroxysulfosuccinimid (Sulfo-NHS) zugegeben wurde, durch die Aminogruppe daran gebunden ist; und
    eine Substanz daran gebunden ist, die selektiv an ein Target binden kann, das fluoreszenzmarkiert werden soll.
  5. Fluoreszenzmarkerstoff gemäß Anspruch 4, wobei die Substanz, die selektiv an ein Target binden kann, ein Antikörper, ein Enzym, ein Lektin oder eine Nukleinsäure ist.
  6. Verwendung des Fluoreszenzmarkerstoffs gemäß Anspruch 4 oder 5, die die Verwendung einer Tumorzelle, einer Leukämiezelle, einer virusinfizierten Zelle oder einer normalen Zelle,
    eines Proteins,
    eines Enzyms oder
    einer Nukleinsäure
    als Target umfasst.
EP09823469.3A 2008-10-30 2009-10-09 Fluoreszenzmarkierungsmaterial und fluoreszenzmarkierungsmittel Not-in-force EP2343535B1 (de)

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